Neuregulin-1 down-regulates expression of ErbB3 expression in hepatoma cell lines at the level of transcription

Background and aims:

Previous studies from our group showed that HCV reduces the expression of ErbB3 in its host cell. As a result of this, the expression of EGFR, which is crucial for the viral entry, is enhanced, which suggests that HCV uses this rearrangement of the ErbB family members to improve its ability to enter the cell. Further investigations identified the ligand of ErbB3 receptor Neuregulin-1 to be responsible for its down-regulation and consequently triggering this circuit. The aim of the present study was to analyze whether down-regulation of ErbB3 by Nrg-1 also occurs in other human hepatoma cell lines and cells of other species like primary mouse hepatocytes and to assess whether this occurs at a transcriptional or post-transcriptional level.

Methods:

HepG2 cells were treated with human Nrg-1 and primary mouse hepatocytes with murine Nrg-1 at different concentrations and different times. The amount of ErbB3 mRNA in both cell types as well as the amount of pre-mRNA in HepG2 cells were measured by RT-PCR. Total protein lysates were analyzed by immunoblot with specific antibodies for ErbB3. Furthermore HepG2 cells were treated with Nrg-1 after adding specific inhibitors of the lysosomal and the proteasomal degradation system.

Results:

The Nrg-1 mediated down-regulation of ErbB3 is an effect which can be also observed in other human hepatoma cell lines, like HepG2. Like in Huh7 cells, the Nrg-1-stimulation caused a reduction of ErbB3 mRNA and protein expression of about 50% after 8h. Also the analysis of pre-mRNA showed a decreased level with a minimum after 4h of stimulation. In primary mouse hepatocytes there is a tendency of Nrg-1 decreasing ErbB3 on transcript level as well. On protein level we cannot show any differences between untreated and treated primary mouse hepatocytes. The inhibition of different pathways of the proteasomal and lysosomal degradation system in Nrg-1 stimulated HepG2 cells showed no alteration of these results so far.

Conclusion:

Evidence is provided that Nrg-1 reduces ErbB3 expression in human hepatoma cell lines, suggesting that this is not an affect, which only holds true for Huh7 cells, preferentially used as model system for HCVcc infection. Furthermore, the reduction of ErbB3 pre-mRNA suggests that the down-regulation occurs on the level of transcription. Concerning the degradation mechanism, we have found no evidence that it is dependent on clathrin-, dynamin- or rapamycin-mediated steps.